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Relishing the view

The wait is over for scientists at St. Jude Children’s Research Hospital who envisioned the day when technology would transform the way they analyze DNA samples. Using new technology that churns out massive amounts of data, investigators now have a comprehensive view of genomes to increase their understanding of cancers and infectious diseases. Known as next-generation sequencing, the ultra high-throughput technology is garnering plenty of buzz in the biotech community and among St. Jude scientists who use the new Genome Sequencing Facility.

“Generally when researchers use traditional sequencing they only look at a gene or a region of interest because it’s difficult and costly to design experiments for unknown regions,” says Caroline Obert, PhD, who oversees the facility. “Next-generation sequencing eliminates this problem and allows researchers to look more thoroughly at the sequence of an organism.”

Building blocks of life

Imagine building a tower out of interlocking building blocks. In sequencing, the blocks are the bases that make up DNA. When the pieces are positioned in a specific order, they can form a variety of organisms. To build a human, 6 billion building blocks are needed; 2 to 4 million are needed for bacteria and approximately 10,000 to 15,000 for viruses.

Sequencing a human is similar to rebuilding a 6 billion-piece tower that has been toppled. The process begins by using parts of the toppled tower that are 200 to 500 blocks long, representing fragmented DNA. The blocks are distributed and locked in place on a special surface called a flow cell, which is coated with adapter sequences that bind the DNA. Loose blocks are used to increase the height of each fragment, with the caveat that certain patterns must be maintained: “T” blocks must be next to “A” blocks and “C” blocks must be paired with “G” blocks. In traditional sequencing, 96 to 384 of these towers, generally 750 bases in length, can be erected simultaneously. In next-generation sequencing, billions of these towers can be built at the same time. However, to compensate for the limited supply of blocks, the towers are fewer than 150 bases high. Every fragment increases one block in height before the next block can be placed. To record this, four pictures, one for each neucleotide, are taken per cycle.

“As researchers, we have important biological questions that we want to answer, diseases that we want to cure, but the technology doesn’t always exist at that moment,” says Michael Dyer, PhD, Developmental Neurobology. “Sometimes it’s worth waiting until the technology comes around. Next-generation sequencing is one of those technologies, and it has really changed the way we think about our biology, our questions and the diseases in which we are interested.”

What’s more attractive is the price tag; the technology is a fraction of the cost of previous sequencing systems.

More data, fewer dollars

Mapping the human genome was too expensive and too massive a project to orchestrate until the NIH launched the Human Genome Project in 1990. It took 13 years, scores of research centers and billions of dollars to complete. Compare that to the two months and less than $2 million it took to sequence DNA pioneer James Watson’s genome using next-generation sequencing. By this year’s end, it will take roughly three weeks and $10,000 to sequence a human genome.

“In the past, it was not cost effective for us to do traditional DNA sequencing on full genomes. Now, we can put 100 flu samples or four bacterial samples into one lane and sequence the genomes for less than $800, allowing our researchers to conduct experiments that they never thought possible,” Obert says.

Elaine Tuomanen, PhD, Infectious Diseases chair, uses the facility to investigate how the Streptococcus pneumoniae genome breaks down into groups that could target different patient populations.

“If you took a snapshot of one person in a crowd, how much would that tell you about the whole crowd? Not much,” Tuomanen says. “In the past, we could ask if individual genes were different between strains and make a guess as to whether that indicated populations differences. Now we can get a true indication of what it takes for this bacteria to cause serious disease in different types of people by sequencing the entire genome of many strains. This shows us amazing differences between strains of the same bacteria that are innocuous versus those that are highly invasive.”

Next-generation sequencing opens a floodgate of opportunities in retinoblastoma research, given the tumor’s historic precedence in cancer genetics.

“We know that every child with retinoblastoma has the RB1 gene mutation, the first-ever human tumor suppressor gene identified,” Dyer says. “However, we know very little about subsequent genetic lesions that drive retinoblastoma tumorigenesis followingRB1 gene inactivation. Next-generation sequencing provides us with a unique opportunity to begin to explore other genetic lesions in retinoblastoma that may provide important clues for new therapies.”

Deciphering the details

Since the DNA fragments are at specific locations during sequencing, software can determine which block was added to a given fragment at any point. If there are enough fragments, and a reference for comparison, the tower can be reassembled to produce answers to investigators’ research questions. Any place where the tower deviates from the reference is a potential genetic mutation.

Mining the data is no small feat. Investigators rely on bioinformatic experts from the Hartwell Center and from Information Sciences to help decipher the details.

“In the past, if you could figure out what caused organisms to act in a certain way, you found the proverbial needle in a haystack. Now that we have access to entire genomes, there is a lot more of the haystack to sort through, and our researchers are finding more needles than they previously expected,” Obert says.